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The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection

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ABSTRACT

Verticillium dahliae, a ubiquitous phytopathogenic fungus, forms resting structures, known as microsclerotia that play crucial roles in Verticillium wilt diseases. VdHog1, a mitogen-activated protein kinase (MAPK), controls microsclerotia formation, virulence, and stress response in V. dahliae. In this study, we present detailed evidence that the conserved upstream component of VdHog1, VdPbs2, is a key regulator of microsclerotia formation, oxidative stress and fungicide response and plant virulence in V. dahliae. We identified VdPbs2, homologous to the yeast MAPK kinase Pbs2. Similar to the VdHog1 deletion mutant, VdPbs2 deletion strains exhibited delayed melanin synthesis and reduced formation of microsclerotia. When exposed to stresses, VdPbs2 mutants were more sensitive than the wild type to osmotic agents and peroxide, but more resistant to inhibitors of cell wall synthesis and some fungicides. Finally, VdPbs2 deletion mutants exhibited reduced virulence on smoke tree and tobacco seedlings. When taken together, we implicate that VdPbs2 and VdHog1 function in a cascade that regulates microsclerotia formation and virulence, but not all VdHog1 dependent functions are VdPbs2 regulated. This study thus provides novel insights into the signal transduction mechanisms that regulate microsclerotia formation and pathogenesis in this fungus.

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Loss of VdPbs2 leads to reduced microsclerotia formation. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2 and ΔVdHog1 grown on PDA for 8 days. The inset shows colony from the opposite view. (B) Microsclerotia formation of the individual strain on cellulose membrane placed onto basal medium plates, and incubated at 25°C at 3, 5, and 24 days. Conidia from each strain were sprayed on the cellulose membrane at a concentration of 105 conidia/ml. (C) Microscopic observation of microsclerotia formation of the above four strains at 24 dpi. Scale bar = 1 mm. (D) Melanized area fractions in the colony were counted by ImageJ. Asterisk indicates significant difference at P < 0.01.
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Figure 1: Loss of VdPbs2 leads to reduced microsclerotia formation. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2 and ΔVdHog1 grown on PDA for 8 days. The inset shows colony from the opposite view. (B) Microsclerotia formation of the individual strain on cellulose membrane placed onto basal medium plates, and incubated at 25°C at 3, 5, and 24 days. Conidia from each strain were sprayed on the cellulose membrane at a concentration of 105 conidia/ml. (C) Microscopic observation of microsclerotia formation of the above four strains at 24 dpi. Scale bar = 1 mm. (D) Melanized area fractions in the colony were counted by ImageJ. Asterisk indicates significant difference at P < 0.01.

Mentions: To investigate the role of VdPbs2 in microsclerotia formation, we first paid our attention to the connection between VdPbs2 function and axenic growth on plate media. Similar to the ΔVdHog1 mutant, ΔVdPbs2 mutants exhibited no significant difference in growth rate but delayed to form microsclerotia on PDA compared with the wild type (Figure 1A). Few melanized microsclerotia can form in the ΔVdPbs2 mutant; by contrast, abundant melanized microsclerotia were produced in the wild type and the ΔVdPbs2/Pbs2 strain (Figure 1A). To determine the influence of VdPbs2 on microsclerotia in detail, we observed the microsclerotia formation on BM. The wild type and the ΔVdPbs2/Pbs2 strain started to accumulate a small amount of melanized microsclerotia at 3 dpi, however, a small number of melanized microsclerotia were observed in the ΔVdPbs2 mutant at 7 dpi (Figure 1B). Furthermore, at 24 dpi, the ΔVdPbs2 and ΔVdHog1 strains still had significant defects in microsclerotia formation, and the melanized area fraction of each strain revealed the deficiency in the melanin accumulation in ΔVdPbs2 and ΔVdHog1 mutants when compared with wild type and the ΔVdPbs2/Pbs2 strain (Figures 1C,D). Strikingly, the melanized microsclerotia were significantly less in the ΔVdHog1 mutant than that of in the ΔVdPbs2 mutant (Figure 1), indicating that VdHog1 may play a more prominent role in the formation of melanized microsclerotia.


The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection
Loss of VdPbs2 leads to reduced microsclerotia formation. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2 and ΔVdHog1 grown on PDA for 8 days. The inset shows colony from the opposite view. (B) Microsclerotia formation of the individual strain on cellulose membrane placed onto basal medium plates, and incubated at 25°C at 3, 5, and 24 days. Conidia from each strain were sprayed on the cellulose membrane at a concentration of 105 conidia/ml. (C) Microscopic observation of microsclerotia formation of the above four strains at 24 dpi. Scale bar = 1 mm. (D) Melanized area fractions in the colony were counted by ImageJ. Asterisk indicates significant difference at P < 0.01.
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Related In: Results  -  Collection

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Figure 1: Loss of VdPbs2 leads to reduced microsclerotia formation. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2 and ΔVdHog1 grown on PDA for 8 days. The inset shows colony from the opposite view. (B) Microsclerotia formation of the individual strain on cellulose membrane placed onto basal medium plates, and incubated at 25°C at 3, 5, and 24 days. Conidia from each strain were sprayed on the cellulose membrane at a concentration of 105 conidia/ml. (C) Microscopic observation of microsclerotia formation of the above four strains at 24 dpi. Scale bar = 1 mm. (D) Melanized area fractions in the colony were counted by ImageJ. Asterisk indicates significant difference at P < 0.01.
Mentions: To investigate the role of VdPbs2 in microsclerotia formation, we first paid our attention to the connection between VdPbs2 function and axenic growth on plate media. Similar to the ΔVdHog1 mutant, ΔVdPbs2 mutants exhibited no significant difference in growth rate but delayed to form microsclerotia on PDA compared with the wild type (Figure 1A). Few melanized microsclerotia can form in the ΔVdPbs2 mutant; by contrast, abundant melanized microsclerotia were produced in the wild type and the ΔVdPbs2/Pbs2 strain (Figure 1A). To determine the influence of VdPbs2 on microsclerotia in detail, we observed the microsclerotia formation on BM. The wild type and the ΔVdPbs2/Pbs2 strain started to accumulate a small amount of melanized microsclerotia at 3 dpi, however, a small number of melanized microsclerotia were observed in the ΔVdPbs2 mutant at 7 dpi (Figure 1B). Furthermore, at 24 dpi, the ΔVdPbs2 and ΔVdHog1 strains still had significant defects in microsclerotia formation, and the melanized area fraction of each strain revealed the deficiency in the melanin accumulation in ΔVdPbs2 and ΔVdHog1 mutants when compared with wild type and the ΔVdPbs2/Pbs2 strain (Figures 1C,D). Strikingly, the melanized microsclerotia were significantly less in the ΔVdHog1 mutant than that of in the ΔVdPbs2 mutant (Figure 1), indicating that VdHog1 may play a more prominent role in the formation of melanized microsclerotia.

View Article: PubMed Central - PubMed

ABSTRACT

Verticillium dahliae, a ubiquitous phytopathogenic fungus, forms resting structures, known as microsclerotia that play crucial roles in Verticillium wilt diseases. VdHog1, a mitogen-activated protein kinase (MAPK), controls microsclerotia formation, virulence, and stress response in V. dahliae. In this study, we present detailed evidence that the conserved upstream component of VdHog1, VdPbs2, is a key regulator of microsclerotia formation, oxidative stress and fungicide response and plant virulence in V. dahliae. We identified VdPbs2, homologous to the yeast MAPK kinase Pbs2. Similar to the VdHog1 deletion mutant, VdPbs2 deletion strains exhibited delayed melanin synthesis and reduced formation of microsclerotia. When exposed to stresses, VdPbs2 mutants were more sensitive than the wild type to osmotic agents and peroxide, but more resistant to inhibitors of cell wall synthesis and some fungicides. Finally, VdPbs2 deletion mutants exhibited reduced virulence on smoke tree and tobacco seedlings. When taken together, we implicate that VdPbs2 and VdHog1 function in a cascade that regulates microsclerotia formation and virulence, but not all VdHog1 dependent functions are VdPbs2 regulated. This study thus provides novel insights into the signal transduction mechanisms that regulate microsclerotia formation and pathogenesis in this fungus.

No MeSH data available.


Related in: MedlinePlus